Abstract Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. One of the most common indications for probiotic treatment is the prevention of antibiotic-associated diarrhea (AAD). Unfortunately, the efficacy of many probiotic products used for AAD is not supported by rigorous independent research, and non-evidence-based clinical usage is common. Data from several studies suggest antibiotic-induced disruption of commensal colonic bacteria results in a significant reduction in short chain fatty acid (SCFA) production and a concomitant reduction in Na-dependent fluid absorption, ultimately resulting in AAD. The probiotic strain Bifidobacterium animalis subsp. lactis BB-12 (BB-12) has been shown to ameliorate a variety of gastrointestinal disease states and is known to produce acetate ? the most abundant primary colonic SCFA ? at concentrations of up to 50 mM in vitro. Thus, we hypothesize that the concurrent administration of BB-12 with antibiotics will protect against the development of AAD by the ability of BB-12 to both generate acetate directly, and increase other SCFAs through cross-feeding of certain bacteria in the Firmicutes phylum. For example, Clostridium, Eubacterium and Roseburia use acetate to produce butyrate, another common SCFA. The primary aim of the R61 phase (N=60) is to determine if BB-12 can mitigate antibiotic-induced reduction in SCFA concentration, as reflected in fecal acetate levels. We hypothesize that antibiotics will decrease fecal SCFAs, but BB-12 supplementation will protect against antibiotic-induced SCFA reduction, and/or be associated with quicker restoration to baseline SCFA levels as compared to control. Antibiotic administration also lowers total microbial counts and diversity in the gut microbiota, disrupting the homeostasis of the gut ecosystem and allowing colonization by pathogens. The secondary aim uses 16S rDNA profiling to determine if BB-12 inhibits antibiotic-induced disruption of the gut microbiota. We hypothesize that antibiotics will diminish the overall number and diversity of bacterial species present in the fecal microbiota, and concurrent BB-12 supplementation will minimize antibiotic-induced shifts in the microbiota, and/or will be associated with shorter recovery to baseline microbiota composition as compared to control. In the R33 phase (N=108), to further delineate the effects of BB-12 administration on the antibiotic-depleted gut microbiota, we will evaluate the timing of probiotic administration in four randomly assigned groups: 1) BB-12 yogurt consumed at the same time as the antibiotic; 2) control yogurt consumed at the same time as the antibiotic; 3) BB-12 yogurt consumed four hours after the antibiotic; and 4) control yogurt consumed four hours after the antibiotic. Our long-term goal is to determine the impact of BB-12 on a variety of gastrointestinal disease states and ages. Elucidation of the mechanism(s) of action will be integral in shaping the direction of future translational research on probiotic effectiveness.